RAPID REGULATION OF PDE-2 AND PDE-4 CYCLIC-AMP PHOSPHODIESTERASE ACTIVITY FOLLOWING LIGATION OF THE T-CELL ANTIGEN RECEPTOR ON THYMOCYTES -ANALYSIS USING THE SELECTIVE INHIBITORS ERYTHRO-9-(2-HYDROXY-3-NONYL)-ADENINE (EHNA) AND ROLIPRAM

The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enz ymes provide the major, detectable cyclic AMP phosphodiesterase activi ties in murine thymocytes. In the absence of cyclic GMP, PDE4 activity predominated (similar to 80% total) but in the presence of low (10 mu M) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (similar to 80% total). The PDE4 selective inh ibitor rolipram dose-dependently inhibited thymocyte PDE4 activity (IC 50 similar to 65 nM). PDE2 was dose-dependently activated (EC(50) simi lar to 1 mu M) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-n onyl)-adenine (EHNA) (IC50 similar to 4 mu M). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies u sing separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as w ell as human PDE2 and PDE4 enzymes. EHNA completely ablated the abilit y of cyclic GMP to activate PDE2 activity, whilst having a much smalle r inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operat ive effects were seen in the absence of cyclic GMP with Hill coefficie nts of similar to 0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) the re was a transient decrease (similar to 83%) in PDE-4 activity and in PDE2 activity (similar to 40%). Both anti-CD3 and anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was follow ed by a sustained and profound increase in activity. In contrast to th at observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular conce ntrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expect ed to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities m ay be rapidly and differentially regulated following ligation of diffe rent cell surface receptors.